Compositions comprising cannabidiol and second therapeutic agents for the treatment of cancer

ABSTRACT

The invention provides synergistic combinations of cannabidiol (CBD) and a second therapeutic agent, such as one or more ChEH/AEBS inhibitors, a naphthoquinone or a derivative thereof, or any combination thereof, effective for the treatment cancer. Compositions containing same and methods of use of same are described.

FIELD OF INVENTION

This invention is directed to combinations of cannabidiol and a secondtherapeutic agent effective in the treatment of cancer. The secondtherapeutic agent includes one or more ChEH/AEBS inhibitors, anaphthoquinone or a derivative thereof, or combinations thereof.

BACKGROUND OF THE INVENTION

Cannabidiol (CBD), a major non-psychoactive constituent of Cannabis, isconsidered an anti-neoplastic agent on the basis of its in-vitro andin-vivo activity against tumor cells. Due to the lack of psychotropicactivity, its pharmacology and therapeutic potential has been underintensive investigation. Recent studies have demonstrated that CBDpossesses a variety of intriguing pharmacological activities, includingimmunosuppressive, anti-inflammatory, anti-convulsive, anxiolytic,anti-psychotic, neuro protective and anti-nausea effects. Itstherapeutic potential has been further substantiated by the recentapproval in Canada of a cannabinoid based medicine containingapproximately equal amounts of Δ9-tetrahydrocannabinol (THC) and CBD,for alleviating neuropathic pain associated with multiple sclerosis.

Cancers are known to affect many areas of the body with the most commontypes of cancers including: cancer of the bile duct, cancer of thebladder, cancer of the bone, cancer of the bowel (including cancer ofthe colon and cancer of the rectum), cancer of the brain, cancer of thebreast, cancer of the neuroendocrine system (commonly known as acarcinoid), cancer of the cervix, cancer of the eye, cancer of theesophagus, cancer of the head and neck (this group includes carcinomasthat start in the cells that form the lining of the mouth, nose, throat,ear or the surface layer covering the tongue). Kaposi's sarcoma, cancerof the kidney, cancer of the larynx, leukemia, cancer of the liver,cancer of the lung, cancer of the lymph nodes, Hodgkin's lymphoma,non-Hodgkin's lymphoma, melanoma, mesothelioma, myeloma, cancer of theovary, cancer of the pancreas, cancer of the penis, cancer of theprostate, skin cancer, soft tissue sarcomas, cancer of the spinal cord,cancer of the stomach, testicular cancer, cancer of the thyroid, cancerof the vagina, cancer of the vulva and cancer of the uterus.

Conventional cancer treatment options are often limited by toxicity oracquired resistance, and novel agents are needed. Cannabidiol (CBD), isa potent, natural compound with reported activity on many cancer types.CBD belongs to the cannabinoid family, a group of pharmacologicallyactive compounds that bind to specific G-protein-coupled receptors.Phytocannabinoids are plant-derived products from Cannabis sativa;endogenous cannabinoids are made in animal and human tissues; andsynthetic cannabinoids are laboratory produced. The G protein-coupledreceptor CBI is found mainly in the brain and nervous system, whereasCB2 is expressed predominantly by immune cells. Recent data suggest thatsome cannabinoids also elicit signal through the vanilloid receptor,whereas others may function in a receptor independent manner.Cannabinoids can modulate signaling pathways central to the growth andspread of cancer. They inhibit cell-cycle progression and chemotaxis,and block angiogenesis. Recent studies have shown that cannabinoids alsoinduce autophagic cell death. Δ9-tetrahydrocannabinol (THC) is one ofthe best-characterized cannabinoids; however, its therapeuticapplications are limited by its psychoactive effects.

There have been some reports of the utility of CBD combination therapywith selective estrogen receptor modulators (SERMs), but there is onlylimited information as to the true utility of this treatment regimen.

There remains a need for new cancer therapies that are more effectiveand are effective against other cancer types than the available agentsand therapies to date.

In one embodiment, the present invention provides a compositioncomprising cannabidiol (CBD) and a second therapeutic agent, effectivefor the treatment of cancer. In one aspect, the second therapeutic agentis a naphthoquinone or a derivative thereof. In another aspect, thesecond therapeutic agent is a ChEH/AEBS inhibitor compound. In someembodiments, the second therapeutic agent is one or more of anaphthoquinone and/or derivative a thereof, one or more ChEH/AEBSinhibitor compounds, or any combination thereof. The compositions areenvisioned for use in the treatment of cancer, and in some embodiments,treatment of estrogen receptor negative cancers are envisioned and insome embodiments, estrogen receptor positive cancers are envisioned.

In one embodiment this invention provides compositions comprisingcombinations of cannabidiol and at least one second therapeutic agent,which is part of the class of ChEH/AEBS inhibitors and uses of same inthe treatment of cancer.

ChEH/AEBS inhibitors comprise different pharmacological classes ofnatural or synthetic compounds. Cholesterol epoxide hydrolase (ChEH)catalyzes the hydration of cholesterol-5,6-epoxides (5,6-EC) intocholestane-3β,5α,6β-triol. ChEH is a hetero-oligomeric complex calledthe microsomal anti-estrogen binding site (AEBS) comprising3bhydroxystetnol-D8-D7-isomerase (D8D7I) and3b-hydroxysterol-D7-reductase (DHCR7). D8D7I and DHCR7 regulatecholesterol biosynthesis and tumor cells growth differentiation, deathand cancer progression.

ChEH/AEBS inhibitors comprise a number of compounds, including, interalia, tesmilifene (DPPE,N,N0-diethylamino-4-(phenylmethylphenoxy)-ethanamine,HCl)3 PBPE, PCPE,MBPE, MCPE, PCOPE, MCOPE, MCOCH2PE; sigma receptor ligands such asSR31747A, BD10008, Haloperidol, SR-31747A, Ibogaine, AC-915, Rimcazole,Trifluoroperazine, Amiodarone; cholesterol biosynthesis inhibitors suchas Triparanol, Terbinafine, U-18666A, Ro 48-8071, AY9944, SKF-525A;unsaturated fatty acids such as oleic acid, α-linolenic acid,acidarachidonic acid (ARA), docosahexaenoic acid (DHA); ring Boxysterols such as 6-Ketocholestanol, 7-Ketocholestanol,7-Ketocholesterol, 7α-hydroxycholesterol, 7β-Hydroxycholesterol,6-Keto-5 hydroxycholestanol, Cholestane-3β,5α,6β-triol(CT) and others aswill be appreciated by the skilled artisan (see for example,Silvente-Poirot S, Poirot M. Cholesterol epoxide hydrolase and cancer.Current opinion in pharmacology. 2012:12(6):69-703; de Medina P,Paillasse M R. Segala G. Poirot M, Silvente-Poirot S:Identification andpharmacological characterization of cholesterol-5,6-epoxide hydrolase asa target for tamoxifen and AEBS ligands. Proc Natl Acad Sci USA 2010,107:13520-13525, all of which are herby incorporated in their entiretyby reference herein).

In another embodiment, the second therapeutic agent is naphthoquinone ora derivative thereof.

The combination therapy/compositions of this invention are active via anestrogen receptor independent mechanism and in some embodiments, theinvention specifically contemplates compositions for use in treatingestrogen receptor negative cancers/tumors.

According to this aspect, and in some embodiments, the ChEH/AEBSinhibitors for use in the described combination therapy may includeselective estrogen receptor modulators (SERMs), which contain a cationicaminoethoxy side chain, such as clomiphene, tamoxifen,4-hydroxy-tamoxifen, raloxifene, Nitromiphene, Ru 39,411 butspecifically excludes non-cationic antiestrogens, such as Faslodex,ICI-164,384 and RU-58.

The compositions of this invention were shown in the Examples, aspresented herein to possess anticancer activity, when multiple classesof ChEH/AEBS inhibitors were used (Example 1).

The compositions of this invention were shown in the Examples, aspresented herein to possess anticancer activity as well, for example,antileukemic activity, and same is clearly mediated via an estrogenreceptor-independent mechanism, as combination treatment with clomipheneand CBD inhibited the CCRF-CEM line, which does not contain an estrogenreceptor.

According to this aspect, with reference to estrogen receptor freecancers/tumors, in one embodiment, the ChEH/AEBS inhibitor is atriphenylethylene (TPE) or a derivative thereof. In another embodiment,the triphenylethylene derivative is selected from the group comprising:antiestrogens (AEs), clomiphene (CL), and tamoxifen (Tam), or acombination thereof.

This invention provides a composition comprising a synergisticcombination of cannabidiol (CBD) and at least one ChEH/AEBS inhibitorcompound, which ChEH/AEBS inhibitor compound is not a SERM. In someembodiments, the ChEH/AEBS inhibitor compound is a selective inhibitorof ChEH/AEBS, and in some embodiments, the selective inhibitor ofChEH/AEBS is PBPE or tesmilifene (DPPE).

In some embodiments, the inhibitor is a cholesterol biosynthesisinhibitor, which in some embodiments, is Triparanol, Teribinafine orU-18666A, or combinations thereof.

In some embodiments, the inhibitor compound is a ring B oxysterol, whichin some embodiments, is 6-ketocholestanol, 7-ketocholestanol,7-ketocholesterol and Cholestane-3b,5a,6b-triol(CT) or combinationsthereof.

In some embodiments, the inhibitor compound is an unsaturated fattyacid, which in some embodiments, is oleic acid, arachidonic acid (ARA)or docosahexaenoic acid (DHA) or combinations thereof.

In some embodiments, the the inhibitor compound is naphthoquinone or aderivative thereof. In some embodiments, the compound is menadione or aderivative thereof.

This invention provides a composition comprising a synergisticcombination of cannabidiol (CBD) and at least one ChEH/AEBS inhibitorcompound, for use in treating an estrogen receptor-negative cancer.

According to this aspect, and in some embodiments, the ChEH/AEBSinhibitor compound is a selective estrogen receptor modulators (SERM)that contains a cationic aminoethoxy side chain. In some embodiments,the SERM is clomiphene, tamoxifen, 4-hydroxy-tamoxifen, raloxifene orcombinations thereof. In some embodiments, the SERM is atriphenylethylene (TPE) or a derivative thereof containing a cationicaminoethoxy side chain or combinations thereof.

In another embodiment, the composition of the present invention furthercomprises a pharmaceutically acceptable carrier.

In some embodiments, this invention provides a method of treatingcancer, comprising administering to a subject in need thereof atherapeutically effective amount of a composition as herein described.In some embodiments, the use of a ChEH/AEBS inhibitor possessing SERMactivity is specifically excluded.

In some embodiments, the invention provides for the use of atherapeutically effective amount of a composition as herein described inthe manufacture of a medicament for use in treating cancer.

In some embodiments, this invention provides a method of treatingcancer, said method comprising administering to a subject in needthereof a therapeutically effective amount of a synergistic combinationof cannabidiol (CBD) and a ChEH/AEBS inhibitor, which ChEH/AEBSinhibitor compound is not a SERM.

In some embodiments, this invention provides a method of treatingcancer, said method comprising administering to a subject in needthereof a therapeutically effective amount of a synergistic combinationof cannabidiol (CBD) and at least one of a naphthoquinone or aderivative thereof, or combinations thereof.

In some embodiments, according to this aspect, the cancer is cancer ofthe bile duct, cancer of the bladder, cancer of the bone, cancer of thebowel (including cancer of the colon and cancer of the rectum), cancerof the brain, cancer of the breast, cancer of the neuroendocrine system(commonly known as a carcinoid), cancer of the cervix, cancer of theeye, cancer of the oesophagus, cancer of the head and neck (this groupincludes carcinomas that start in the cells that form the lining of themouth, nose, throat, ear or the surface layer covering the tongue).Kaposi's sarcoma, cancer of the kidney, cancer of the larynx, leukaemia,acute leukemia, chronic lymphocytic leukemia, cancer of the liver,cancer of the lung, cancer of the lymph nodes. Hodgkin's lymphoma,non-Hodgkin's lymphoma, melanoma, mesothelioma, myeloma, cancer of theovary, cancer of the pancreas, cancer of the penis, cancer of theprostate, skin cancer, soft tissue sarcomas, cancer of the spinal cord,cancer of the stomach, testicular cancer, cancer of the thyroid, cancerof the vagina, cancer of the vulva and cancer of the uterus.

In some embodiments, this invention provides a method for treating asubject afflicted with a blood or a bone marrow related cancer in asubject in need thereof comprising administering to said subject atherapeutically effective amount of a synergistic combination ofcannabidiol (CBD) and a ChEH/AEBS inhibitor, which ChEH/AEBS inhibitorcompound is not a SERM.

In some embodiments, this invention provides a method for treating asubject afflicted with a blood or a bone marrow related cancer in asubject in need thereof comprising administering to said subject atherapeutically effective amount of a synergistic combination ofcannabidiol (CBD) and a naphthoquinone or a derivative thereof

In some embodiments, this invention provides a method for treating asubject afflicted with a blood or a bone marrow related cancer in asubject in need thereof comprising administering to said subject atherapeutically effective amount of a composition as herein described.In some embodiments, the composition comprises CBD and a ChEH/AEBSinhibitor, and in some embodiments, the composition comprises CBD and anaphthoquinone or a derivative thereof and in some embodiments, thecomposition comprises CBD and combinations of a ChEH/AEBS inhibitorand/or a naphthoquinone or a derivative thereof. In some embodiments,the use of a ChEH/AEBS inhibitor possessing SERM activity isspecifically excluded.

In some embodiments, this invention provides a method for treating asubject afflicted with glioblastoma, in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a synergistic combination of cannabidiol (CBD) and a ChEH/AEBSinhibitor. In some embodiments, the glioblastoma is estrogen receptornegative. In some embodiments, the glioblastoma is estrogen receptorpositive and the ChEH/AEBS inhibitor compound is not a SERM.

In some embodiments, this invention provides a method for treating asubject afflicted with glioblastoma, in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a synergistic combination of cannabidiol (CBD) and anaphthoquinone or a derivative thereof.

In some embodiments, this invention provides a method for treating asubject afflicted with glioblastoma, in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a composition as herein described. In some embodiments, theuse of a ChEH/AEBS inhibitor possessing SERM activity is specificallyexcluded.

In some embodiments, this invention provides a method for treating asubject afflicted with breast cancer, in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a synergistic combination of cannabidiol (CBD) and a ChEH/AEBSinhibitor. In some embodiments, the breast cancer is estrogen receptornegative. In some embodiments, the breast cancer is estrogen receptorpositive and the ChEH/AEBS inhibitor compound is not a SERM.

In some embodiments, this invention provides a method for treating asubject afflicted with breast cancer, in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a synergistic combination of cannabidiol (CBD) and anaphthoquinone or a derivative thereof.

In some embodiments, this invention provides a method for treating asubject afflicted with breast cancer, in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a composition as herein described. In some embodiments, thecomposition comprises CBD and a ChEH/AEBS inhibitor, and in someembodiments, the composition comprises CBD and a naphthoquinone or aderivative thereof and in some embodiments, the composition comprisesCBD and combinations of a ChEH/AEBS inhibitor and/or a naphthoquinone ora derivative thereof. In some embodiments, the use of a ChEH/AEBSinhibitor possessing SERM activity is specifically excluded.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graphically depicts the synergistic effect of cannabidiol withDPPE on inhibition of growth of HL-60 cell line.

FIG. 2. Graphically depicts the synergistic effect of cannabidiol withDPPE on inhibition of growth of CCRF-CEM cell line.

FIG. 3. Graphically depicts the synergistic effect of cannabidiol with7-ketocholesterol on inhibition of growth of HL-60 cell line.

FIG. 4. Graphically depicts the synergistic effect of cannabidiol with7-ketocholesterol on inhibition of growth of CCRF-CEM cell line.

FIG. 5. Graphically depicts the synergistic effect of cannabidiol withtriparanol on inhibition of growth of HI-60 cell line.

FIG. 6. Graphically depicts the synergistic effect of cannabidiol withtriparanol on inhibition of growth of CCRF-CEM cell line.

FIG. 7. Graphically depicts the lack of synergistic effect ofcannabidiol with ICI 182,780 on inhibition of growth of Hl-60 cell line.

FIG. 8. Graphically depicts the lack of synergistic effect ofcannabidiol with ICI 182,780 on inhibition of growth of CCRF-CEM cellline.

FIG. 9A Graphically depicts the effect of cannabidiol on viability ofHL-60 cells incubated with different concentrations of cannabidiol andvehicle in 5% FCS serum and serum free medium for 24 h, 10% FCS mediumfor 24 and 48 h.

FIG. 9B Graphically depicts the effect of cannabidiol on viability ofCCRF-CEM cells incubated with different concentrations of cannabidioland vehicle in serum free and 5% serum containing medium for 24 h.

FIG. 9C Graphically depicts the effect of cannabidiol on viability ofMCF-7 cells incubated with different concentrations of cannabidiol andvehicle in 5% serum containing medium for 24 h. Cell viability wasdetermined by XTT assay.

FIG. 9D Graphically depicts the effect of cannabidiol on viability ofA-172 cells incubated with different concentrations of cannabidiol andvehicle in 5% serum containing medium for 48 h. Cell viability wasdetermined by XTT assay.

FIG. 10A Graphically depicts the effect of clomiphene on viability ofHL-60 cells incubated with different concentrations of clomiphene andvehicle in 5% serum containing medium for 24 h. Cell viability wasdetermined by XTT assay.

FIG. 10B Graphically depicts the effect of clomiphene on viability ofCCRF cells incubated with different concentrations of clomiphene andvehicle in 5% serum containing medium for 24 h. Cell viability wasdetermined by XTT assay.

FIG. 10C Graphically depicts the effect of clomiphene on viability ofA-172 cells were incubated with different concentrations of clomipheneand vehicle in 5% serum containing medium for 48 h. Cell viability wasdetermined by XTT assay.

FIG. 11A Graphically depicts the effect of tamoxifen on viability ofHL-60 cells incubated with different concentrations of tamoxifen andvehicle in 5% serum containing medium for 24 h. FIG. 11B Graphicallydepicts the effect of tamoxifen on viability of CCRF cells incubatedwith different concentrations of tamoxifen and vehicle in 5% serumcontaining medium for 24 h.

FIG. 11C Graphically depicts the effect of tamoxifen on viability ofMCF-7 cells incubated with different concentrations of tamoxifen andvehicle in 5% serum containing medium for 24 h. Cell viability wasdetermined by XTT assay.

FIG. 12A graphically depicts the synergistic effect of cannabidiol withclomiphene, tamoxifen and menadione (Mena) on growth inhibition of HL-60cells incubated with different concentrations of cannabidiol, clomipheneand vehicle in 5% FCS medium for 24 h.

FIG. 12B graphically depicts the synergistic effect of cannabidiol withclomiphene, tamoxifen and menadione (Mena) on growth inhibition of HL-60cells incubated with different concentrations of cannabidiol, tamoxifenand vehicle in 5% FCS medium for 24 h of HL-60 cells incubated withdifferent concentrations of cannabidiol, tamoxifen and vehicle in 5% FCSmedium for 24 h.

FIG. 12C graphically depicts the synergistic effect of cannabidiol withclomiphene, tamoxifen and menadione (Mena) on growth inhibition of CCRFcells incubated with different concentrations of cannabidiol, menadioneand vehicle in 5% FCS medium for 24 h. Cell viability was determined byXTT assay.

FIG. 13 graphically depicts the additive effect of cannabidiol withdoxorubicin on growth inhibition of HL-60 cells incubated with differentconcentrations of cannabidiol, doxorubicin (Dox) and vehicle in 5 and10% FCS medium for 24 h. Cell viability was determined by XTT assay.

FIG. 14A graphically depicts the synergistic effect of cannabidiol withclomiphene and tamoxifen, on growth inhibition of CCRF-CEM cellsincubated with different concentrations of cannabidiol, clomiphene andvehicle in 5% FCS medium for 24 h. Cell viability was determined by XTTassay.

FIG. 14B graphically depicts the synergistic effect of cannabidiol withclomiphene and tamoxifen, on growth inhibition of CCRF-CEM cellsincubated with different concentrations of cannabidiol, tamoxifen andvehicle in 5% FCS medium for 24 h.

FIG. 15A graphically depicts the synergistic effect of cannabidiol withtamoxifen, on growth inhibition of MCF-7 cells incubated with differentconcentrations of cannabidiol, tamoxifen and vehicle in 5% FCS mediumfor 24 h. Cell viability was determined by XTT assay.

FIG. 15B graphically depicts the synergistic effect of cannabidiol withtamoxifen, on growth inhibition of MCF-7 cells pre-treated with 10 nMestradiol for 30 min, then incubated with different concentrations ofcannabidiol, tamoxifen and vehicle in 5% FCS medium for 24 h. Cellviability was determined by XTT assay.

FIG. 16 graphically depicts the synergistic effect of cannabidiol withtamoxifen, on growth inhibition of MCF-7 cell line. The A-172 cells wereincubated with different concentrations of cannabidiol, clomiphene andvehicle in 5% FCS medium for 48 h. Cell viability was determined by XTTassay.

FIG. 17A graphically depicts the induction of apoptosis in HL-60 cellsby cannabidiol, clomipheneand menadione. Dose-response effect ofcannabidiol, clomiphene and menadioneon induction of apoptosis in HL-60cells incubated with cannabidiol and vehicle in 5% FCS medium for 24 and48 h is shown.

FIG. 17B graphically depicts the induction of apoptosis in HL-60 cellsby cannabidiol, clomipheneand menadione. Dose-response effect ofcannabidiol, clomiphene and menadioneon induction of apoptosis in HL-60cells were incubated with cannabidiol, clomiphene and vehicle in 5% FCSmedium for 24 h is shown.

FIG. 17C graphically depicts the induction of apoptosis in HL-60 cellsby cannabidiol, clomipheneand menadione. Dose-response effect ofcannabidiol, clomiphene and menadioneon induction of apoptosis in HL-160cells were incubated with cannabidiol, menadione and vehicle in 5% FCSmedium for 24 h is shown.

FIG. 18A graphically depicts the induction of apoptosis in CCRF-CEMcells by cannabidiol, clomiphene and tamoxifen in CCRF-CEM cellsincubated with cannabidiol and vehicle in serum free and 5% FCS mediumfor 24 h.

FIG. 18B graphically depicts the induction of apoptosis in CCRF-CEMcells by cannabidiol, clomiphene and tamoxifen in CCRF-CEM cellsincubated with cannabidiol, clomiphene and vehicle in 5% FCS medium for24 h.

FIG. 18C graphically depicts the induction of apoptosis in CCRF-CEMcells by cannabidiol, clomiphene and tamoxifen in CCRF-CEM cells wincubated with cannabidiol, tamoxifen and vehicle in 5% FCS medium for24 h.

FIG. 19 is a micrograph depicting the morphological features of normalHL-60 cells and apoptotic cells were visualized by fluorescencemicroscopy. Original magnification X400 in cells treated with CBD for 24h

FIG. 20 depicts the effect of cannabidiol and clomiphene on downregulation of Mcl-1 in CCRF-CEM cells treated with 20p M cannabidiol forthe indicated time points. Cells were collected and washed two timeswith PBS, then cell lysates were prepared using RIPA buffer. Proteinswere subjected to SDS-PAGE and immunoblotted with antibodies againstMCI-1 and calreticulin (CRN).

FIG. 21A graphically depicts the effect of cannabidiol, clomiphene andcannabidiol+clomiphene on tumor volume in a mouse xenograft modeltransplanted with HL-60 cells. Tumor volumes were measured once per weekby caliper (n=6). (*: P<0.05; **: P<0.01).

FIG. 21B depicts the effect of cannabidiol, clomiphene andcannabidiol+clomiphene on tumor volume in a mouse xenograft modeltransplanted with HL-60 cells, where tumors were harvested and therepresentative images of tumors in each group are shown.

FIG. 22A graphically depicts the effect of cannabidiol and clomiphene onAML primary cells incubated with and without and clomiphene for 24 h.Cell viability was measured by XTT reduction.

FIG. 22B graphically depicts the effect of cannabidiol and clomiphene onCLL primary cells incubated with and without and cannabidiol for 24 h.Cell viability was measured by XTT reduction.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention is a composition comprisingcannabidiol (CBD) and at least one ChEH/AEBS inhibitor effective in thetreatment of malignancies such as, but not limited to blood cancer, bonemarrow related cancer, breast cancer and glioblastoma.

This invention provides a composition comprising cannabidiol (CBD) andat least one naphthoquinone or a derivative thereof effective in thetreatment of malignancies such as, but not limited to blood cancer, bonemarrow related cancer, breast cancer and glioblastoma.

The term “cannabidiol” (CBD) as used herein refers to aphyto-cannabinoid produced from the plat Cannabis species. In someembodiments CBD used in the present invention is in a purified form. Inother embodiments, CBD is a component of a plant extract. In someembodiments, a plant extract comprises at least 10% to 95% CBD. In someembodiments, a plant extract comprises at least 20% to 80% CBD. In someembodiments, a plant extract comprises at least 30% to 70% CBD. In someembodiments, a plant extract comprises at least 40% to 60% CBD.

In another embodiment, CBD is a botanical drug substance (BDS). A“botanical drug substance” or “BDS” is defined in the Guidance forIndustry Botanical Drug Products Draft Guidance, August 2000, USDepartment of Health and Human Services, Food and Drug AdministrationCentre for Drug Evaluation and Research as: “A drug derived from one ormore plants, algae, or microscopic fungi. It is prepared from botanicalraw materials by one or more of the following processes: pulverisation,decoction, expression, aqueous extraction, ethanolic extraction or othersimilar processes.” A botanical drug substance does not include a highlypurified or chemically modified substance derived from natural sources.

In another embodiment, where a synthetic CBD is used the term isintended to include compounds, metabolites or derivatives thereof, andpharmaceutically acceptable salts of CBD.

In another embodiment, the composition of the present inventioncomprises chemically-modified derivatives of fully-decarboxylated CBDwhich retain desired activity, or more preferably natural derivativesexhibiting improved activity which are produced according to standardprinciples of medicinal chemistry. In some embodiments,fully-decarboxylated CBD derivatives may exhibit a lesser degree ofactivity than the starting material so long as they retain sufficientactivity to be therapeutically effective or exhibit improvements inproperties desirable in pharmaceutically active agents such as improvedsolubility, enhanced uptake or reduced toxicity.

In another embodiment, the composition of the present inventioncomprises a naphthoquinone or a derivative thereof. Naphthoquinones area class of organic compounds derived from naphthalene. Non-limitingexamples of naphthoquinones are: 1,2-Naphthoquinone, 1,4-Naphthoquinone,menadione, 2,6-Naphthoquinone, Hexahydroxy-1,4-naphthalenedione,5-hydroxy-1,4-naphthalenedione, 2-Methoxy-1,4-naphthoquinone,Pentahydroxy-1,4-naphthalenedione and2,3,5,7-Tetrahydroxy-1,4-naphthalenedione.

The term “ChEH/AEBS inhibitor” as used herein refers to compounds asherein described, and as known in the art to be identified as same.

In some aspects, the ChEH/AEBS inhibitors comprise differentpharmacological classes of natural or synthetic compounds. Cholesterolepoxide hydrolase (ChEH) catalyzes the hydration ofcholesterol-5,6-epoxides (5,6-EC) into cholestane-3β,5α,6β-triol. ChEHis a hetero-oligomeric complex called the microsomal anti-estrogenbinding site (AEBS) comprising 3bhydroxysterol-D8-D7-isomerase (D8D7I)and 3b-hydroxysterol-D7-reductase (DHCR7). D8D7I and DHCR7 regulatecholesterol biosynthesis and tumor cells growth differentiation, deathand cancer progression.

ChEH/AEBS inhibitors comprise a number of compounds, including, interalia, tesmilifene (DPPE,N,N0-diethylamino-4-(phenylmethylphenoxy)-ethanamine.HCl)3 PBPE, PCPE,MBPE, MCPE, PCOPE, MCOPE, MCOCH2PE, sigma receptor ligands such asSR31747A, BD10008 Haloperidol, SR-31747A, Ibogaine, AC-915, Rimcazole,Trifluoroperazine, Amiodarone, cholesterol biosynthesis inhibitors suchas Triparanol, Terbinafine, U-18666A, Ro 48-8071, AY9944, SKF-525A,unsaturated fatty acids such as oleic acid, α-linolenic acid,acidarachidonic acid (ARA), docosahexaenoic acid (DHA), ring Boxysterols such as 6-Ketocholestanol, 7-Ketocholestanol,7-Ketocholesterol, 7α-hydroxycholesterol, 7β-Hydroxycholesterol,6-Keto-5 hydroxycholestanol, Cholestane-3β,5α,6β-triol(CT) and others aswill be appreciated by the skilled artisan (see for example,Silvente-Poirot S. Poirot M. Cholesterol epoxide hydrolase and cancer.Current opinion in pharmacology. 2012; 12(6):696-703; de Medina P.Paillasse M R, Segala G. Poirot M, Silvente-Poirot S:Identification andpharmacological characterization of cholesterol-5,6-epoxide hydrolase asa target for tamoxifen and AEBS ligands. Proc Natl Acad Sci USA 2010,107:13520-13525, all of which are herby incorporated in their entiretyby reference herein).

In some aspects, in particular, with reference for compositions, methodsand uses for treating estrogen receptor negative cancer, the ChEH/AEBSinhibitors comprise a triphenylethylene (TPE) derivative such as theantiestrogens (AEs) clomiphene (CL) and tamoxifen (Tam).

In another embodiment, the term “cancer” is blood cancer. In anotherembodiment, the term “cancer” is bone marrow cancer or bone marrowrelated cancer. In another embodiment, the term “cancer” is breastcancer. In another embodiment, the term “cancer” is glioblastoma. Inanother embodiment, blood cancer is leukemia. In another embodiment,blood cancer is acute promyelocytic leukemia (AML). In anotherembodiment, blood cancer is myeloblastic leukemia. In anotherembodiment, blood cancer is Non-Hodgkin's lymphoma. In anotherembodiment, blood cancer is Myeloma. In another embodiment, blood canceris lymphoma. In another embodiment, bone marrow cancer is aMyeloproliferative disorder.

In some embodiments, the composition of the present invention is usedfor treating a subject afflicted with cancer. In some embodiments,treating a subject afflicted with cancer using the composition of thepresent invention induces a synergistic effect as compared to thecombined effect of using each of the compounds alone. A synergisticeffect is a coordinated or correlated action of two or more compounds,so that the combined action is greater than the sum of each compoundacting separately. Non-limiting examples of compounds of the presentinvention that have a synergistic effect when co-administered with CBDare DPPE, 7-ketocholesterol, triparanol, or combinations thereof. Insome embodiments, the non-limiting examples of compounds of the presentinvention that have a synergistic effect when co-administered with CBDin estrogen receptor negative cancers include triphenylethylene (TPE)derivatives such as the antiestrogens (AEs) clomiphene (CL) andtamoxifen (Tam). In some embodiments, the synergistic effect whenco-administered with CBD includes the naphthoquinone derivativemenadione.

In other embodiments, treating a subject afflicted with cancer with acombination of CBD and ChEH/AEBS inhibitors and/or a naphthoquinone or aderivative thereof induces more than an additive effect as compared tothe combined effect of administration of each of the compounds alone.The term additive effect means that the combined action of two or morecompounds is equal to the sum of each compound acting separately.

Referring to Example 1, and FIGS. 1-6, representative ChEH/AEBSinhibitors were demonstrated to possess anti-cancer activity, whetherselective ChEH/AEBS inhibitors (DPPE) or, for example, ring B oxysterols(7-ketocholesterol) or a cholesterol biosynthesis class of inhibitors(triparanol) was used, when used in combination with CBD, demonstratingtrue synergistic effect.

Referring to Example 2, a representative naphthoquinone or a derivativethereof, menadione was shown to possess anti-cancer activity, as well,when used in combination with CBD, demonstrating true synergisticeffect.

Examples of additional compounds that have an additive effect whenco-administered with CBD are doxorubicin and anthracyline as shown inFIG. 13.

In some embodiments, the synergistic effect of the composition of thepresent invention in treating cancer is at least 1.1 fold higher, thanthe additive effect in treating cancer by administering the compounds ofthe same composition separately.

In some embodiments, the synergistic effect in treating cancer using thecomposition of the present invention is between 1.1 fold to 2 foldhigher, 2 fold to 3 fold higher, 3 fold to 4 fold higher, 4 fold to 5fold higher than the additive effect in treating cancer by administeringthe compounds of the same composition separately.

In some embodiments, the synergistic effect in treating cancer using thecomposition of the present invention is more than 5 fold higher than theadditive effect in treating cancer by administering the compounds of thesame composition separately.

In some embodiments, the composition of the present invention is acombination of CBD and an ChEH/AEBS inhibitors, wherein the molar ratioof the CBD to the ChEH/AEBS inhibitors is between 50:1 to 1:50 (CBD:theChEH/AEBS inhibitors). In some embodiments, the composition of thepresent invention is a combination of CBD and a naphthoquinone or aderivative thereof, wherein the molar ratio of the CBD to thenaphthoquinone or a derivative thereof is between 50:1 to 1:50 (CBD:thenaphthoquinone or a derivative thereof).

In some embodiments, the composition of the present invention is acombination of CBD and an ChEH/AEBS inhibitors, wherein the molar ratioof the CBD to ChEH/AEBS inhibitors is between 30:1 to 1:30(CBD:ChEH/AEBS inhibitors).

In some embodiments, the composition of the present invention is acombination of CBD and a naphthoquinone or a derivative thereof, whereinthe molar ratio of the CBD to the naphthoquinone or a derivative thereofis between 30:1 to 1:30 (CBD:the naphthoquinone or a derivativethereof).

In some embodiments, the composition of the present invention is acombination of CBD and an ChEH/AEBS inhibitors, wherein the molar ratioof the CBD to ChEH/AEBS inhibitors is between 10:1 to 1:10(CBD:ChEH/AEBS inhibitors).

In some embodiments, the composition of the present invention is acombination of CBD and a naphthoquinone or a derivative thereof, whereinthe molar ratio of the CBD to the naphthoquinone or a derivative thereofis between 10:1 to 1:10 (CBD:the naphthoquinone or a derivativethereof).

In some embodiments, the composition of the present invention is acombination of CBD and an ChEH/AEBS inhibitors, wherein the molar ratioof the CBD to naphthoquinone is between 5:1 to 1:5 (CBD:ChEH/AEBSinhibitors).

In some embodiments, the composition of the present invention is acombination of CBD and a naphthoquinone or a derivative thereof, whereinthe molar ratio of the CBD to the naphthoquinone or a derivative thereofis between 5:1 to 1:5 (CBD:the naphthoquinone or a derivative thereof)

In some embodiments, the composition of the present invention is acombination of CBD and an ChEH/AEBS inhibitors, wherein the molar ratioof the CBD to ChEH/AEBS inhibitors is between 2:1 to 1:2 (CBD:ChEH/AEBSinhibitors).

In some embodiments, the composition of the present invention is acombination of CBD and a naphthoquinone or a derivative thereof, whereinthe molar ratio of the CBD to the naphthoquinone or a derivative thereofis between 2:1 to 1:2 (CBD:the naphthoquinone or a derivative thereof).

In some embodiments, the composition of the present invention is acombination of CBD and an ChEH/AEBS inhibitor, wherein the molar ratioof the CBD to ChEH/AEBS inhibitors is 1:1 (CBD:ChEH/AEBS inhibitors).

In some embodiments, the composition of the present invention is acombination of CBD and a naphthoquinone or a derivative thereof, whereinthe molar ratio of the CBD to the naphthoquinone or a derivative thereofis 1:1 (CBD:the naphthoquinone or a derivative thereof).

In another embodiment, the composition of the present invention isformulated as a pharmaceutical composition further comprising one ormore pharmaceutically acceptable carriers, expedients or diluents.

In some embodiments, the term “treatment” as used herein refers to anyresponse to, or anticipation of, a medical condition in a mammal,particularly a human, and includes but is not limited to: preventing themedical condition from occurring in a subject, which may or may not bepredisposed to the condition, but has not yet been diagnosed with thecondition and, accordingly, the treatment constitutes prophylactictreatment for the medical condition; inhibiting the medical condition,e.g., arresting, slowing or delaying the onset, development orprogression of the medical condition; or relieving the medicalcondition, e.g., causing regression of the medical condition or reducingthe symptoms of the medical condition.

In another embodiment, the term “subject” refers to a human afflictedwith cancer. In another embodiment, the term “subject” refers to amammal such as a pet or a farm animal afflicted with cancer.

In another embodiment, the term “administering” as used herein, includesdelivery of a composition or one or more pharmaceutically activeingredients to a subject, by any appropriate methods, which serve todeliver the composition or its active ingredients or otherpharmaceutically active ingredients to the subject. In anotherembodiment, the method of administration may vary depending on variousfactors, such as for example, the components of the pharmaceuticalcomposition or the nature of the pharmaceutically active or inertingredients, the site of the potential or actual malady, age andphysical condition of the subject. Some non-limiting examples of ways toadminister the composition of the present invention to a subjectinclude: oral, intravenous, topical, intrarespiratory, intraperitoneal,intramuscular, parenteral, sublingual, transdermal, intranasal, aerosol,intraocular, intratracheal, intrarectal, vaginal, dermal patch, eyedrop, ear drop or mouthwash.

As used herein, “therapeutically effective amount” refers toadministration of an amount of the composition of the present invention,to a subject in need thereof that achieves prevention, inhibition orregression of the cancer in the subject in need thereof.

In some embodiments, the amount of CBD in the composition administeredto the subject is between 0.1 mg/kg (body weight)/day and 50 mg/kg (bodyweight)/day. In some embodiments, the amount of CBD in the compositionof the present invention is between 10 mg/kg (body weight)/day and 1000mg/kg (body weight)/day. In some embodiments, the amount of CBD in thecomposition of the present invention is between 50 mg/kg (bodyweight)/day and 500 mg/kg (body weight)/day. In some embodiments, theamount of CBD in the composition of the present invention is between 500mg/kg (body weight)/day and 2000 mg/kg (body weight)/day.

In some embodiments, the amount of ChEH/AEBS inhibitor or inhibitors, orthe amount of naphthoquinone or a derivative thereof in the compositionadministered to the subject is between 0.1 mg/kg (body weight)/day and50 mg/kg (body weight)/day. In some embodiments, the amount of ChEH/AEBSinhibitor inhibitor or inhibitors, or the amount of naphthoquinone or aderivative thereof in the composition of the present invention isbetween 10 mg/kg (body weight)/day and 1000 mg/kg (body weight)/day. Insome embodiments, the amount of ChEH/AEBS inhibitor inhibitor orinhibitors, or the amount of naphthoquinone or a derivative thereof inthe composition of the present invention is between 50 mg/kg (bodyweight)/day and 500 mg/kg (body weight)/day. In some embodiments, theamount of ChEH/AEBS inhibitor or inhibitors, or the amount ofnaphthoquinone or a derivative thereof in the composition of the presentinvention is between 500 mg/kg (body weight)/day and 2000 mg/kg (bodyweight)/day.

In another embodiment, the duration of the treatment is between 24 h to14 days. In another embodiment, the duration of the treatment is between24 h to 30 days. In another embodiment, the duration of the treatment isbetween 1 to 12 months. In another embodiment, the method is used totreat a subject with a chronic cancer and the duration of the treatmentis for the life time of the subject.

In some embodiments, administration of the composition of the presentinvention to a subject afflicted with cancer induces apoptosis of acancerous cell thereby treating the subject. Apoptosis of cancer cellsis quantified by methods known in the art such as, but no limited to theXTT assay described below in the materials and methods section.

In some embodiments, administration of the composition of the presentinvention to a subject afflicted with cancer suppresses tumor growththereby treating the subject. In some embodiments, suppression of tumorgrowth refers to the slowing or prevention of growth in the size of atumor after administration of the composition of the present invention.In some embodiments, tumor growth is compared to relevant clinical dataof the treated cancer as known in the art. In some embodiments, tumorgrowth is compared to pre-treatment tumor size and/or volume of thetreated subject. Measurement of the size of a tumor is done by methodsknown in the art.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes 1-111 Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York: Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis.J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait. M. J., ed.(1984); “Nucleic Acid Hybridization” Hames. B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317. Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990): Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Materials and Methods

Reagents: Reagents were purchased from commercial vendors, dissolved in100% DMSO and stored at −20° C. The final DMSO concentration was 0.1%.Acridinc orange/ethidium bromide stain was prepared at a concentrationof 1 mg/ml in PBS. XTT and all other chemicals were acquired from Sigma(St. Louis, Mo.). Control cultures contained the vehicle, which had noeffect by itself.

Cell culture: human HL-60, myeloblastic leukemia and CCRF-CEM, acutelymphoblastic leukemia cell line were cultured in RPMI-1640 supplementedwith 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 100U/ml penicillin and 10 μg/ml streptomycin. MCF-7, breast cancer andA-172, glioblastoma cell lines were cultured in DMEM supplemented with10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin. Cultures were maintained at 37°C. in a humidified atmosphere with 5% CO2 and maintained in exponentialphase by transfer to fresh medium every 2-3 days. Experiments wereconducted in serum free medium and 5% or 10% FCS medium. The trypanbluedye exclusion method [17] was used for cell counting performed using ahemocytometer under light microscope.

Determination of cell viability: cell viability analysis of HL-60,CCRF-CEM, A-172 and MCF-7 cells was assessed by their (2,3[-bis-2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxianilide,inner salt (XTT) reduction activity. 100 μl of 2.5×10⁵ cells/ml wasincubated with treatments at the indicated time. At the end of theincubation period, 25 μl of 1 mg·ml XTT solution (containing 0.2 mMphenazinemethosulphate (PMS) was added and the cells were incubated foran additional 1 h. The OD values were measured using an ELISA reader at450 nm with a reference wavelength of 650 nm. Data were expressed as themean percentage of the three replicates, normalized to the untreatedvehicle.

Morphological quantification of apoptosis: for determination ofapoptosis, cells were harvested (650 g, 7 min) and stained with acridineorange/ethidium bromide at a final concentration of 0.05 mg/ml. Thismethod allows distinguishing between live, necrotic and apoptotic cells(FIG. 11). Cells were scored as alive if their nuclei exhibited normalmorphology and were green. Cells exhibiting normal morphology and orangecolor were indicated as necrotic. Cells were scored as apoptotic iftheir nuclei exhibited condensation of the chromatin and/or nuclearfragmentation. At least 100 cells were counted under a fluorescencemicroscope and the percentage of affected cells was calculated.

Western blot analysis: for Western blot analysis, 5×10⁷ cells/ml wastreated with and without 20 μM CBD and 20 μM clomiphene (CU for theindicated periods of time. Cells washed twice with ice cold PBS andlysed in RIPA buffer (50 mM Tris-HCl, 0.1% NP-40, 1 mM DTT, 0.25Msucrose, 2 mM MgCl₂, pH 7.4) for 30 min on ice. After centrifugation,the post-nuclear supernatant containing the cell soluble fraction wereloaded on to a 12% SDS-PAGE gel. After electrophoresis, the gels wereblotted onto a PVDF membrane (Bio-Rad. Hercules, Calif.), blocked with5% (w/v) milk for overnight at 4° C., temperature, washed briefly inTris-Buffered Saline Tween-20 (TBST), and then probed overnight at 4°C., with the anti-mouse human anti-MCI-1 and calreticulin (CRN) primaryantibodies. Primary antibody binding was detected with anti-mouse IgGconjugated to horseradish peroxidase (Jackson Immunoresearch, Avondale,Pa.), and made visible by enhanced chemiluminescence (ECL) (BiologicalIndustries, Israel), according to manufacturer's instructions.Antibodies against MCl-1 and calreticulin (CRN) were purchased fromSanta Cruz, Calif., USA.

In vivo anti-tumor efficacy of CBD and CL: NOD/SCID mice were bred andhoused at animal facility of Ben-Gurion University of the Negev, Israel.All animals were used between 7-8 weeks old of age. For induction oftumor, mice were irradiated with 2.7 Gy X ray radiation for 2 min. Thefollowing 5 hours, mice were injected with 1 million viable HL-60 cellson right flank of mice. Mice were monitored for tumorigenesis. Tumorswere measured during 14 days and their volumes (mm³) were calculated as(d2XD)/2 (where d is the shortest and D is the longest diameter of thetumor in mm). Following the day mice were divided into four groups (6mice/group): vehicle (veh), cannabidiol group (CBD group), clomiphene(CL group), and combination (CBD+CL group). The mice were injectedperitmorally with 5 mg CBD/kg for CBD group, 10 mg CL/kg for CL groupand 5 mg CBD+10 mg CL for CBD+CL group (dissolved in 0.1 ml of sterilePBS supplemented with 5 mg/ml defatted and dialyzed bovine serumalbumin) or its vehicle. The injection was repeated once a day, 5 daysper week, and tumor volumes were checked twice a week until the vehicledied and the remained animals were sacrificed.

Data Presentation and Statistical Analysis: Viability experiments wereperformed in triplicates and experiments were repeated at least two orthree times. Where statistical analysis was performed, same wasevaluated by Students t-test. Some of the data is presented in theFigures provided, with statistically significant differences designatedas *p<0.05, **p<0.01.

Example 1 Combinations of Cannabidiol and ChEH/AEBS Inhibitors Cause aReduction in Cancer Cell Viability

The effect of combinations of cannabidiol and ChEH/AEBSinhibitors-exposure on the viability of HL-60 and CCRF-CEM, A-172 andMCF-7 cell lines in vitro was examined. To this end, the tumor cellswere cultured in 10% FCS supplemented medium and exposed to variousconcentrations of cannabidiol (1, 5, 10, and 30 μM) for 48 h in 5% FCScontaining medium and 1, 5, 10 and 30 μM ChEH/AEBS inhibitors (FIGS. 1,2, 3 and 4).

With regard to the HL-60 assays, the results showed that exposure tocannabidiol at concentrations of even 1 μM or more when in combinationwith ChEH/AEBS inhibitors for 48 h treatment led to a significantreduction in the number of viable cells. The reduction in cell viabilitywas dose responsive having a profound effect on reduction of cellviability even at very low concentrations 1 μM or 5 μM.

Similarly, results of the combination of exposure to cannabidiol atconcentrations of even 1 μM or more when in combination with ChEH/AEBSinhibitors in the estrogen receptor deficient CCRF-CEM cell line showeda significant reduction in the number of viable cells. The reduction incell viability was dose responsive having a profound effect on reductionof cell viability even at very low concentrations 1 μM or 5 μM of each.

These results demonstrate that representative ChEH/AEBS inhibitors,whether selective inhibitors (DPPE) or, for example, ring B oxysterols(7-ketocholesterol), when provided in combination with CBD exhibitpotent anticancer activity.

To further elaborate on the fact that CBD combination therapy with broadChEH/AEBS inhibitor use is effective, another representative ChEH/AEBSinhibitor was evaluated for its activity in combination with CBD againstHL-60 and CCRF-CEM viability (FIGS. 5 and 6). In this case, an ChEH/AEBSinhibitor of the cholesterol biosynthesis class of inhibitors(triparanol) was selected and used in concentrations of 30, 60, 100 and200 μM. Although higher concentrations of the triparanol were evaluatednonetheless, a significant reduction in the number of viable cells, withthe optimum triparanol concentration of 10 μM with 1-10 μM of CBD.

FIGS. 7 and 8 provide the results of the negative control for thesestudies, combination therapy of CBD and with ICI-182,780 (1, 5, 10 and30 μM) a non cationic antiestrogen whose combination with CBD providesno added benefit.

Thus, representative classes of ChEH/AEBS inhibitors when provided as acombination therapy with CBD exhibited profound anti-cancer activity.

Example 2 Cannabidiol, TPEs (Clomiphene and Tamoxifen) and MenadioneCause a Reduction in Cell Viability

The effect of cannabidiol-exposure on the viability of HL-60 andCCRF-CEM, A-172 and MCF-7 cell lines in vitro was examined. To this end,the rumor cells were cultured in 10% FCS supplemented medium and exposedto various concentrations of cannabidiol (5, 10, 15, 20 and 30 μM) for24 h and 48 h in 10% for 5% FCS containing medium and 0.01, 0.03, 0.1,0.3, 1, 3 and 5 μM CBD for 24 h in serum free medium (FIGS. 9A, 9B, 9Cand 9D). The results showed that exposure to cannabidiol atconcentrations of 5 μM or more for 24 h and 48 h treatment led to asignificant reduction in the number of viable cells (FIGS. 9A, 9B, 9Cand 9D). The reduction in cell viability was dose responsive both at 24and 48 hr exposure (FIGS. 9A, 9B, 9C and 9D). CBD had a profound effecton reduction of cell viability even at very low concentration 0.2 μM inserum free condition for 24 h of exposure. IC₅₀ value was obtained in10% FCS medium after 48 h as 16 μM. With decreased serum concentration(5% FCS) in the medium, IC₅₀ was also obtained in 24 h. In serum freemedium IC₅₀ was 0.26 μM for 24 h. In 10% FCS medium cells proved to beless sensitive to CBD for 24 h. The statistical significance of cellviability reduction was P<0.05.

The effect of clomiphene and tamoxifen on HL-60, CCRF-CEM, A-172 andMCF-7 cell lines was also examined. To this end the dose dependentreduction in cell viability was observed with clomiphene in 5% serumsupplemented medium for 24 h, for HL-60, CCRF-CEM and 48 hr for A-172cell line (FIGS. 10 and 11). The IC 50 values for clomiphene in HL-60,A-172 and CCRF-CEM cell lines were found to be 14, 11 and 15 μMrespectively. The IC 50 values of tamoxifen for HL-60, CCRF-CEM andMCF-7 was found to be 12, 16 and 12 μM respectively.

Example 3 Synergistic Effect of CBD with TPE and Naphthoquinone

In this study, CBD had synergistic anticancer effects with clomiphene,tamoxifen and menadione (FIG. 12 A, B, C). As shown in FIG. 4 thecombination CBD, clomiphene, tamoxifen and menadione effectively reducedthe viability of HL-60 cells. The most effective combinations were foundas 5 μM CBD and 15 μM clomiphene (FIG. 12A), 10 μM CBD and 10 μMtamoxifen (FIG. 12B). Menadione and CBD showed more than additive inreducing the cell viability at high concentration of menadione (FIG.12C). Conventional chemotherapeutic drug anthracycline, doxorubicinshowed additive effect with CBD in reduction in viability of HL-60 cells(FIG. 13).

The effect of CBD and TPE (clomiphene and tamoxifen) in estrogenreceptor (ER) negative cell lines CCRF-CEM, A-172 and ER positive cellline MCF-7 was examined. As shown in FIGS. 14A and B CBD with TPEscaused synergistic reduction in the viability of CCRF-CEM cellssimilarly to HL-60 cell line. This finding supports the notion that thecannabidiol and TPE interact independently of ER involvement inreduction of cell viability mechanism. The most potent combination wasfound as 10 μM CBD and 10 μM tamoxifen which synergistically reduced theviability of MCF-7 cells (FIG. 15). The human glioblastoma cell line,A-172 cell viability was also found to be reduced synergistically withCBD and clomiphene for 48 hr of exposure in 5% serum medium (FIG. 16).

To evaluate the role of ER involvement in cell viability of MCF-7 withthe combination of CBD and tamoxifen, p estradiol was added 30 minbefore the drug treatment in order to block the ER. Interestingly therewas no significant difference in cell viability between D estradioltreated and untreated groups (FIGS. 15A and 15B). Thus these resultsstrongly showed that these drugs are acting independent of ER inreduction of cell viability.

Example 4 Nuclear Morphological Changes are Characteristics of Apoptosis

The effect of CBD on apoptosis of cancer cell lines was examined. CBDshowed dose and time dependent induction of apoptosis in both HL-60 andCCRF-CEM cell lines (FIG. 17A and FIG. 18A). The combination of CBD withTPEs had a synergistic effect in induction of apoptosis whereas withmenadione, CBD showed more than additive effect in induction ofapoptosis of HL-60 cell line (FIGS. 17B and 17C). Similar results werealso found with CCRF-CEM cell line (FIG. 18). A representativemicrograph is shown in FIG. 19.

Example 5 Down-Regulation of Mcl-1 with CBD

Over expression of Mcl-1 is associated with survival of leukemic cells;CBD down regulated the Mcl-1 expression in CCRF-CEM cells. CBD showedthe down regulation of Mcl-1 by 3 h of treatment (FIG. 20).

Example 6 Inhibition of Tumor Growth in a Mouse Xenograft ModelTransplanted with HL-60 Cells

Treatment of the xenografted mice with 5 mg/kg CBD, 10 mg/kg CL aloneand combination resulted in significant suppression of HL-60 tumorgrowth from days 7 through 14 (FIG. 21).

Example 7 Effect of CBD and Clomiphene on AML and CLL Primary Cells

The cytotoxic effect of CBD and clomiphene on AML and CLL primary cellswas evaluated, as well. 5 μM clomiphene and 10 μM CBD synergisticallyinduced cell death within 24 hr (FIG. 22).

1-55. (canceled)
 56. A method of treating cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising: a synergistic combination ofcannabidiol (CBD) and at least one cholesterol epoxidehydrolase/anti-estrogen binding site (ChEH/AEBS) inhibitor compound,wherein the ChEH/AEBS inhibitor compound is a selective estrogenreceptor modulator (SERM); and a pharmaceutically acceptable carrier.57. The method of claim 56, wherein the SERM comprises wherein the SERMcomprises a cationic aminoethoxy side chain.
 58. The method of claim 56,wherein the cancer is cancer of the bile duct, cancer of the bladder,cancer of the bone, cancer of the bowel, cancer of the brain, cancer ofthe breast, cancer of the neuroendocrine system, cancer of the cervix,cancer of the eye, cancer of the esophagus, cancer of the head and neck,Kaposi's sarcoma, cancer of the kidney, cancer of the larynx, leukemia,acute leukemia, chronic lymphocytic leukemia, cancer of the liver,cancer of the lung, cancer of the lymph nodes, Hodgkin's lymphoma,non-Hodgkin's lymphoma, melanoma, mesothelioma, myeloma, cancer of theovary, cancer of the pancreas, cancer of the penis, cancer of theprostate, skin cancer, soft tissue sarcomas, cancer of the spinal cord,cancer of the stomach, testicular cancer, cancer of the thyroid, cancerof the vagina, cancer of the vulva and cancer of the uterus.
 59. Themethod of claim 56, wherein the cancer is a blood or a bone marrowrelated cancer.
 60. The method of claim 56, wherein the cancer isglioblastoma.
 61. The method of claim 56, wherein the cancer is breastcancer.
 62. The method of claim 56, wherein the cancer is an estrogenreceptor-negative cancer.
 63. The method of claim 57, wherein the SERMcomprises clomiphene, tamoxifen, 4-hydroxy-tamoxifen, raloxifene or acombination thereof.
 64. The method of claim 63, wherein the SERMcomprises clomiphene.
 65. The method of claim 57, wherein the SERMcomprises a triphenylethylene (TPE) or a derivative thereof containing acationic aminoethoxy side chain, or a combination thereof.
 66. Acomposition comprising a synergistic combination of cannabidiol (CBD)and at least one cholesterol epoxide hydrolase/anti-estrogen bindingsite (ChEH/AEBS) inhibitor compound, which ChEH/AEBS inhibitor compoundis not a selective estrogen receptor modulator (SERM) and is a selectiveinhibitor of ChEH/AEBS, wherein the CBD is present in an amountsufficient to provide a dose of 0.1 mg/kg to 50 mg/kg to a subject andthe ChEH/AEBS inhibitor compound is present in an amount sufficient toprovide a dose of 500 mg/kg to 2000 mg/kg to the subject, and theselective inhibitor is 1-[2-[4-(phenylmethyl)phenoxy]ethyl]-Pyrrolidine(PBPE) or tesmilifene (DPPE).
 67. The composition of claim 66, whereinthe selective inhibitor is DPPE.
 68. The composition of claim 66,further comprising a pharmaceutically acceptable carrier.
 69. Ananti-cancer pharmaceutical composition which comprises the compositionof claim 68 in a therapeutically effective amount.
 70. A method oftreating leukemia, breast cancer, or glioblastoma, which comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the composition of claim
 68. 71. A method for treating asubject in need of leukemia, breast cancer, or glioblastoma therapy,which comprises administering to the subject a therapeutically effectiveamount of the composition of claim 66 for the treatment of leukemia,breast cancer, or glioblastoma.
 72. The method of claim 71, wherein theselective inhibitor is DPPE.
 73. The method of claim 71, wherein thecomposition is administered intravenously, orally, or sublingually. 74.The method of claim 71, wherein the composition is administered to thesubject for a duration of between 24 hours to 14 days, or between 24hours to 30 days.
 75. The composition of claim 66, wherein the ChEH/AEBSinhibitor comprises a cholesterol biosynthesis inhibitor.
 76. Thecomposition of claim 75, wherein the cholesterol biosynthesis inhibitorcomprises Triparanol, Teribinafine, U-18666 A, or a combination thereof.77. The composition of claim 66, wherein the ChEH/AEBS inhibitorcomprises a ring B oxysterol.
 78. The composition of claim 77, whereinthe ring B oxysterol comprises 6-ketocholestanol, 7-ketocholestanol,7-ketocholesterol and Cholestane-3b,5a,6b-triol(CT) or combinationsthereof.
 79. A composition comprising a synergistic combination ofcannabidiol (CBD) and at least one naphthoquinone or a derivativethereof.
 80. The composition of claim 79, wherein the naphthoquinone ora derivative thereof comprises menadione.